The article by Liu et al. (1), on immune and metabolic effects of antigen-specific immunotherapy via multiple β-cell–specific peptides in type 1 diabetes, uses a popular method to estimate the number of regulatory T cells (Tregs) in peripheral blood that has been shown to have defects (2,3). Essentially, by considering as Tregs all CD3+CD4+CD25highCD127low/− cells, one unavoidably includes many non-Tregs in this population. The “high” and “low/−” intensities of CD25 and CD127, respectively, are subjective because of a continuum of intensity values in these parameters (e.g., see Supplementary Fig. 1 of Liu et al. [1]). A more accurate estimation of effector/active Tregs and naive/resting Tregs can be achieved via the use of the CD3/CD4/CD25/CD45RA-RO panel, where the former are CD3+CD4+CD25highCD45RA (fraction II) and the latter are CD3+CD4+CD25intCD45RA+ (fraction I), inherently avoiding such subjectiveness (2). The CD127low/− and FoxP3+ populations of CD3+CD4+CD25high cells are not congruent (see Supplementary Fig. 1 of Liu et al. [1]). In fact, there is a 13.5% increase in the FoxP3+ cells compared with the CD127low/− ones (7.70% vs. 6.78%). This is not surprising, as the method of Miyara et al. (2) for Treg delineation identified a FoxP3+ population that lacks regulatory activity (CD3+CD4+CD25intCD45RA; fraction III). By differentially coloring the six fractions in a CD25/CD127 dot plot, we were able to show that demarcation of Tregs via the CD3+CD4+CD25highCD127low/− criterion unavoidably excludes a sizeable portion of naive Tregs but includes many cells from fraction III (non-Tregs) as well as cells from the uncharacterized fraction of CD3+CD4+CD25int/lowCD45RA cells (gray dots in Fig. 3B of Petsiou et al. [3]).

Detailed proteomic/transcriptomic investigation of the properties of five of the six fractions has uncovered the several adaptive mechanisms in cellular signaling by which fractions I and II maintain their Treg identity, while CD4+ T cells from other fractions are activated in the canonical manner via select stimuli (4). Besides identifying and characterizing the properties of at least six CD4+ T-cell fractions, this method holds promise for identifying the fraction(s) bearing (self or foreign) antigen-specific T cells and enumerating their respective specificities via pMHCII tetramer staining (5). This is an important piece of information when dealing with peptide-specific immunotherapy of type 1 diabetes, as Liu et al. do. The monoclonal antibody panel required for this manner of Treg delineation is already part of the large panel that the authors used for immunophenotyping (see Supplementary Table 2 of Liu et al. [1]). Thus, the data are there, they just need to be analyzed in this suggested manner. Last, there is the outstanding issue of the presence of FoxP3+ in the CD4+CD25low T-cell population (otherwise uncharacterized) in the peripheral blood of patients with type 1 diabetes (and from several autoimmune diseases), as cited in Petsiou et al. (3). This, in parallel to the diminution of Tregs from the CD4+CD25high cell population, makes the method of Miyara et al. (2) the method of choice for delineation of CD4+ T cell fractions: two Treg populations as well as variably activated and naive ones. The convenient separation of the CD25 intensity of expression into four scales (negative, low, intermediate, and high) to delineate different CD4+ T-cell fractions should be taken advantage of.

G.K.P. retired from the Technological Educational Institute (TEI) of Epirus, Arta, Greece, on 1 September 2018. The affiliation is given for identification purposes only. As of 1 October 2018, the TEI of Epirus has been absorbed by the University of Ioannina. The respective department is now called the Department of Agriculture.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

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